VEGF gene expression is regulated post‐transcriptionally in macrophages

Abstract

The macrophage is critical to the innate immune response and contributes to human diseases, including inflammatory arthritis and plaque formation in atherosclerosis. Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that is produced by macrophages. To study the regulation of VEGF production in macrophages we show that stimulation of monocyte‐macrophage‐like RAW‐264.7 cells by lipopolysaccharide (LPS) increases expression of VEGF mRNA and protein. Three alternative splicing VEGF mRNA isoforms are produced, and the stability of VEGF mRNA increases following cellular activation. To study post‐transcriptional regulation of the VEGF gene the 3′‐untranslated region (3′ UTR) was introduced into the 3′ UTR of the luciferase gene in a reporter construct. In both RAW‐264.7 cells and thioglycollate‐elicited macrophages, the 3′ UTR sequence dramatically reduces reporter expression. Treatment with activators of macrophages, including LPS, lipoteichoic acid, and VEGF protein, stimulates expression of 3′ UTR reporters. Finally, mapping studies of the 3′ UTR of VEGF mRNA show that deletion of the heterogeneous nuclear ribonucleoprotein l binding site affects basal reporter expression in RAW‐264.7 cells, but does not affect reporter activation with LPS. Together these results demonstrate that a post‐transcriptional mechanism contributes to VEGF gene expression in activated macrophage cells.

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“…Increases in VEGF-A or Arginase-1 levels are generally associated with an M2 macrophage phenotype. 46,59 Overall, these gene expression results are indicative of the emergence of mixed M1/M2 phenotypes in AMs exposed to previously stimulated NCs. However protein level data showed no change in any cytokine investigated (Fig.…”

“…AMs also initially produced significantly higher amounts of VEGF-A mRNA ( p = 0.001) relative to NMs, in agreement with previous literature describing the production of VEGF by RAW 264.7 cells upon LPS stimulation. 59 At the protein level, AMs produced significantly greater amounts of TNF-a (*5.3-fold; p < 0.001) and IL-6 (*8 Fig. S2B).…”

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“…These results contrast with those originating from other cell populations. For instance, VEGF gene expression was shown to be regulated by LPS in macrophages (19), and TNF-␣ is a potent activator of VEGF synthesis and secretion by neutrophils (22,53). However, the different VEGF isoforms have distinctive biological effects and pattern of expression in specific cell population (29,30).…”

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“…Alternative exon splicing of a single VEGF gene is the basis for this molecular heterogeneity [10] . VEGF 165 is a major component, [25,30] . Post-transcriptional regulation involves mRNA binding proteins acting on cis elements to alter mRNA stability or the efficiency of translation [30] .…”

“…The half-life (t 1/2 ) of the VEGF mRNA was calculated from the slope of the logarithmic plot of relative mRNA levels versus time of decay, using a linear regression analysis [25] . Statistical analyses All data were expressed as means± SD.…”